The art and science of cell and tissue culture has evolved continuously since 1897, when Loeb demonstrated that cells of the blood and connective tissue can be maintained in a viable state in vitro. Methods have been developed to study various differences in cells and tissues grown in semi-solid media as contrasted with growth in liquid media. Similar studies have been made of differences in growth and function of cells and tissues in liquid which is stirred and in liquid which is quiescent.
Many types of cells and tissues grown in quiescent liquid cultures develop as monolayers, which are less representative of three-dimensional structures for cells and tissues which occur naturally in animals. Investigators studying the effects of chemotherapeutic agents and ionizing radiation on cancer cells have recognized the importance of culturing cells as three-dimensional structures that correspond to micro-lesions in vivo. Extensive basic studies of multicellular growths have shown that cells and tissues grown under conditions which favor three-dimensional structures are more useful than monolayer-grown cells. For example, cells grown as three-dimensional structures may express genes that are silent in monolayer-grown cells. Thus, three-dimensional structures are proving useful in studies of differentiation, immunology and molecular genetics.
U.S. Pat. No. 5,523,228 issued Jun. 4, 1996 to Ingram et al. discloses improved methods and apparatus for generating cell and tissue cultures which have the desired three-dimensional structure. That patent discloses injecting a mixture of liquid culture medium and cells to be grown into a flexible container or bag which is permeable to oxygen and carbon dioxide. The culture medium and cells are introduced into the bag with a hypodermic syringe through a conventional hypodermic syringe port (with a female Luer lock) sealed between two gas-permeable sheets which form the walls of the bag. The bag is completely filled with the mixture and slowly rotated about a horizontal axis to keep the cells suspended under low-shear hydrodynamic flow. Metabolized culture medium is removed and replaced with fresh medium as required from time to time by using a hypodermic syringe connected to the syringe port.
The apparatus disclosed in U.S. Pat. No. 5,523,228 produces cells with the desired three-dimensional structure, but suffers the disadvantage that the hypodermic syringe port introduces an irregularity to the interior surface of the bag, which traps cells and creates excessive localized turbulence, which interferes with three - dimensional cell growth. Moreover, the hypodermic syringe port makes it difficult and time-consuming to remove all air bubbles from the chamber when filling it with liquid culture medium and cells. As explained in the patent, it is important to fill the chamber completely (zero head space) because air bubbles increase hydrodynamic shear and turbulence, which discourages desired three-dimensional growth.